INTERLEUKIN-8 AND MONOCYTE CHEMOTACTIC PROTEIN-1 GENE-EXPRESSION AND PROTEIN-PRODUCTION BY HUMAN ORBITAL FIBROBLASTS

Orbital inflammation is common, but the mechanisms underlying leukocyt ic infiltration of orbital tissue are poorly understood. We studied re sident human orbital fibroblasts (OF) interleukin-8 (IL-8), and monocy te chemotactic protein-1 (MCP-1) mRNA expression and protein secretion in response to lipopolysaccharide (LPS) or recombinant human cytokine s that are present during inflammation. Third-passaged cultured human OF were left unstimulated or incubated with varying concentrations of LPS, recombinant interleukin-1-beta (rIL-1 beta), recombinant tumor ne crosis factor-alpha (rTNF-alpha), or recombinant interferon-gamma (rIF N-gamma) for 2, 4, 8, or 24 h. Northern blot analysis and ELISA were p erformed to determine OFIL-8 and MCP-1 mRNA expression and protein sec retion, respectively. Experiments were performed in triplicate and rep eated four times on different cell lines. OF lacked constitutive IL-8 or MCP-1 gene expression, but produced substantial dose-dependent incr eases in steady-state IL-8 and MCP-1 mRNA expression by 2 h of LPS or cytokine stimulation (rIL-1 beta>rTNF-alpha: >LPS>IFN-gamma), maintain ed at 24 h. ELISA for IL-8 and MCP-1 proteins showed significant time- and dose-dependent OF secretion after exposure to recombinant cytokine or LPS (rIL-1 beta>rTNF-alpha>LPS), measured after 4 h of exposure (p < 0.01). This increased in the media over the next 20 h. rIFN-gamma w as a potent stimulant of OF MCP-1, significant by 2 h (p < 0.05), but only a weak stimulant of IL-8 at 24 h. OF secreted IL-8 and MCP-1 in r esponse to LPS and proinflammatory cytokines, indicating that these re sident cells within the orbit have the capacity to actively participat e in the initiation and propagation of orbital inflammation. Strategie s aimed at modulating local mediators may be helpful in the management of orbital inflammatory disease.